HMG-CoA reductase inhibitor preparation process

ABSTRACT

The invention provides a simple and selective method for the deacylation of 4-acylated statins during the preparation of statins from a fermentation broth, more specifically a reduction of impurities from the process, by increasing the pH of the fermentation broth.

The present invention relates to a process of preparing, purifyingand/or isolating compounds which are hydroxymethyl glutaryl coenzyme A(HMG-CoA) reductase inhibitors (or their precursors) such as lovastatin.The invention in particular relates to purifying such a compound from acomposition comprising microorganisms that have produced it (such as byusing an adsorbent resin), performing an extraction using toluene,performing a lactonization reaction, if necessary (e.g. in the toluene),and washing the toluene with water before isolating the final compound.

It is known that certain mevalonate derivatives are active ashypercholesterolemic agents, and these function by limiting cholesterolbiosynthesis by inhibiting the enzyme HMG-CoA reductase. Mevalonatederivatives include the naturally occurring fungal metaboliteslovastatin and compactin.

Lovastatin can be produced by fermentation of various microorganismsincluding Aspergillus terreus, during which it is produced in a freehydroxy acid form along with a number of by-products. Consequently,isolation usually involves removal of the microorganisms (usuallyreferred to as the biomass) and by-products, followed by lactonization(to the closed ring or lactone form, called lovastatin) and removal ofby-products. Several methods of isolation have been developed, but theyare usually multi-step processes involving extraction from afermentation broth (or a filtrate thereof) using organic solvents. Dueto the number of steps involved the yield of lovastatin is generallylow. In addition, large quantities of potentially hazardous solvents areusually required which not only entails the requisite safety measuresbut additionally increases costs dealing with solvent disposal.

The present invention seeks to provide a process of purifying orisolating an (3-hydroxy-3-methyl glutaryl-coenzyme A) HMG-CoA reductaseinhibitor, such as lovastatin, or a precursor thereof, starting from acomposition comprising cells (e.g. microorganisms) which have producedthe precursor or inhibitor, in good yield and/or using reducedquantities of solvent.

In a first aspect the invention provides a process of purifying acompound, which is preferably an HMG-CoA reductase inhibitor, or aprecursor thereof, of the general formula I or II:

wherein:

each of R¹ and R² independently represents a hydrogen atom, a methylgroup or a hydroxyl group; and

R³ represents a (straight chain or branched) C₂₋₆ alkyl group;

or a salt or isomer thereof;

and there is one double bond present in the first ring, between eithercarbon atoms 3 and 4 or atoms 4 and 4a, and no or one double bond in thesecond ring, which if present is between either carbon atoms 4a and 5 oratoms 5 and 6;

from an (e.g. aqueous) composition comprising cells, such asmicroorganisms, that have produced the compound, the process comprising:

(a) adjusting, if necessary, the pH of the composition to be at least7.5;

(b) removing (e.g. by filtering) from the composition the cells toobtain a solution of the compound (e.g. a filtrate);

(c) contacting the solution with a resin so that the compound isadsorbed onto the resin; and

(d) removing the compound from the resin.

By this process the compound can be absorbed onto a resin and sopurified. The cells can be removed by filtration (in which case one isleft with a filtrate), centrifugation (so one obtains a supernatant) oreven by decantation. Whichever method is employed, the remaining cells(or biomass), or the waste liquid resulting from passage over the resin,can then be discarded, without further processing. This can be achievedbecause no (non-aqueous) solvent need be added to either the compositionor the solution prior to contact with the resin, unlike prior artprocesses. The compound is suitably removed from the resin by elution.

The purification process can thus result in a concentration of thecompound, and when the resin is a hydrophobic one, it is possible toremove polar components present in the original composition.

The adjustment to a pH of at least 7.5 has been found to improvepurification because this assists in the dissolution of the compound.Several prior art processes, such as those described in U.S. Pat. Nos.4,231,938 and 4,294,926 (Merck) purify the precursor (to lovastatin)from an essentially neutral fermentation-derived medium, but that is notbasified, unlike the process of the present invention, and as a resultnot all the precursor has dissolved. Therefore, some of the precursorcan remain either with the microorganisms when they are removed (bycentrifugation).

A precursor is a compound that can be converted into the reductaseinhibitor by a relatively simple or straightforward chemical conversion,such reactions being known in the art. This may involve several steps,although sometimes only one reaction is required, and that islactonization (described later in relation to the second aspect of theinvention). This is the closing of the “open” ring and results in acompound of the formula II. However, many compounds of formula I areinhibitors although they are sold (and often administered) in thelactone form of formula II. These are converted to the open ring form offormula I in vivo.

Alternatively, a precursor can be converted into a reductase inhibitorby a hydroxylation reaction, such as at the 6-position, and this can beachieved by using various microorganisms, for example those described inGB-A-2,077,264 (Sankyo).

Preferred reductase inhibitors are able to inhibit the biosynthesis ofcholesterol, and so can be useful as hypercholesterolemic agents. Thetest for HMG-CoA reductase inhibition is well known in the art, but forexample one can use the methodology of Beg et al, FEBS Letters80:123-129 (1977) or described in J. Biol. Chem. 234:2835 (1959).Suitable enzymes can be prepared as described by Kleinsek et al, PNAS74:1431-5 (1977).

In formulae I and II, suitably R² represents a hydrogen atom. PreferablyR³ represents a 1-methyl propyl group (as is the case for lovastatin,pravastatin and compactin) or a 1,1-dimethyl propyl group (as is thecase with simvastatin).

Preferably the compound will have one double bond in each of the firstand second rings, suitably located between carbon atoms 3 and 4 andcarbon atoms 4a and 5 (as is the case with lovastatin, pravastain,compactin and simvastatin, although compounds which have only one doublebond, located in the first ring, are contemplated (such as betweencarbon atoms 3 and 4, in the case of dihydrolovastatin). Other compoundsthat are contemplated have a double bond in the first ring betweencarbon atoms 4 and 4a and a double bond in the second ring betweencarbon atoms 5 and 6 and are described in GB-A-2,077,264 (Sankyo)

The (usually aqueous) composition comprising the cells (usuallymicroorganisms although plant or animal cells modified to produce theprecursor can be used) can be any composition which comprises thecompound of general formula I or II. It will often contain water andmicroorganisms, particularly when the composition comprises afermentation broth, a sample removed from such a broth (e.g. beforefermentation is complete) or a sample from the broth which has beenstored, for example at a low temperature.

The microorganism can be any microorganism that is capable of producingthe compound and this includes bacteria, yeasts and (preferably) fungi.Preferred microorganisms are fungal, for example of the genusAspergillus, Monascus, Penicillium , Paecilomyces, Hypomyces, Phoma,Pleurotus, Doratmyces, Eupenicillium, Gymnoaxus and Trichoderma.

Of these, one can optimally use a fungus of the species Penicilliumcitrinum or Aspergillus terreus, for example strain AD43.

Preferably, in (a) the composition is adjusted to have a pH of from 9 to13, such as from 10 to 11. The composition may be diluted with water.The pH can be adjusted by any suitable alkali. Preferably, it is analkali metal hydroxide, for example sodium hydroxide, for example 1-3N,such as about 2N, NaOH.

At this stage in the process the composition is usually a fermentationbroth. The composition is adjusted to the desired pH to dissolve thecompound. In some prior art processes, such as the two Merck patentsmentioned above, a solvent is added to the composition, in the hope thatthe compound will dissolve, but the present invention can avoid the needfor such solvent addition. As will be appreciated, if one adds a solventat this stage then it needs to be removed later on in the process. Byavoiding the need for a solvent, one can significantly reduce the amountof contaminated waste considerably.

The cells (or the biomass) can be removed by filtration, centrifugationor decantation. Filtering, if used, can be carried out by any methodknown in the art but is preferably over a rotary vacuum filter or amembrane filter press. The remaining cells (biomass), e.g. in the formof a filter cake can be subsequently washed with water. Preferably it iswashed with at least 3 times its volume of water. One then can obtain asolution of the compound which can be a decant, filtrate or supernatant.

After the removal of the cells in (b) the solution can be furtherbasified and/or heated. Thus the pH may be increased, such as to aboutpH 12. Heating may also be employed. The purpose of this is to convertan unwanted by-product into a useful compound. For example, duringfermentation, such as in the production of lovastatin, O-acetyllovastatin is formed. This by-product can be removed at a later stage,but preferably it can be converted into lovastatin itself, thusincreasing the overall yield of this reductase inhibitor. Thisconversion can be achieved by treating the solution at a high alkalinepH at an elevated temperature.

Thus, the pH of the solution is preferably then adjusted in (a) to from11 to 13 and/or suitably heated. Preferably heating is to at least 50°C. The duration of heating is dependant on the temperature employed. Forexample, the solution can be heated for at least 30 minutes (e.g. atabout 60° C.) or for at least 10 minutes (e.g. at about 90° C.).

The solution is then contacted with a resin in stage (c) that is adaptedto allow the compound to be adsorbed onto it. The solution can be passedover a bed of the resin. This is to concentrate the compound and/orremove polar impurities. The flow rate of the solution can be varied butis preferably from 3 to 6 bed volumes per hour during loading and from 3to 6 bed volumes at any other time. The resin can be any suitable resinbut is preferably a hydrophobic resin, such as a polystyrene resin, forexample Amberlite™ XAD-16 or XAD-1180. Preferably up to 40 g of thecompound is loaded per liter of resin. However, before this the resincan be conditioned, such as with 1 to 6 bed volumes of aqueous alkali,for example 0.02N NaOH. After adsorption (of the compound) the resin canbe washed to remove any impurities which have not been adsorbed. Washingcan be carried out using any suitable liquid, for example anacetone-aqueous sodium hydroxide solution, preferably up to 15 %, e.g.from 5 to 15 % acetone in a (e.g. 0.2N) sodium hydroxide solution.

The compound is then removed from the resin, usually by elution. Thepreferred eluent is a water-miscible solvent, such as an aqueous solvente.g. comprising water and acetone, or a C₁₋₄ alcohol. Suitably thesolvent comprises at least 80%, such as at least 60%, acetone: theremainder is preferably water. Following removal of the adsorbedcompound, the resin can be regenerated and conditioned for a followingcycle by washing. By using a resin the amount of solvent used can be upto 15 times less than for prior art methods.

In a second aspect the invention provides a process (which can followthe process of the first aspect) of purifying or isolating, from anaqueous composition, a compound of general formula I or II, the processcomprising:

(a) adjusting, if necessary, the pH of the composition to be less than7, and optionally adding acetone;

(b) contacting the composition with a water-immiscible solventcomprising toluene so that at least some of the compound is extracted bydissolving in the (toluene-containing) solvent; and

(c) optionally, if the compound is of formula I, lactonizing (e.g. inthe solvent) the compound to give a compound of formula II.

Preferably the compound is of general formula I: on lactonization it canthus yield a compound of general formula II

wherein R¹, R² and R³ are as defined above for formula I and there isone double bond present in the first ring (possessing R²), betweeneither carbon atoms 3 and 4 or atoms 4 and 4a, and no or one double bondin the second ring (possessing R¹), which if present is between eithercarbon atoms 4a and 5 or atoms 5 and 6.

It will be seen therefore that the process of the second aspect can besubsequent to the first aspect. In that event, the aqueous compositionis preferably the eluate resulting from stage (d), or removal of thecompound (e.g. of formula I), from the resin, of the first aspect.

The aim of the second aspect is to further concentrate the compound. Inaddition, it can automatically provide the compound in a solvent that issuitable for lactonization. In several prior art processes, a differentsolvent is used for lactonization than for extraction, and not only doesthis result in an increase in costs, but it reduces yields.

In addition, the second aspect may result in the removal (by extraction)of undesired polar components, in particular those that are polar at apH less than 7.

The pH of the composition is preferably adjusted to be from 3 to 5 andmost preferably to a pH of about 4. The composition is then contactedwith the (toluene-containing) solvent so that the compound is extracted(into the toluene). The amount of toluene added is dependent on thedesired concentration of the compound, which can vary from 0.1 g/l to 10g/l (at room temperature). The duration of contact is not critical,however, preferably the composition is contacted with the solvent for atleast 15 minutes.

The solvent comprises toluene because this has been found to give goodpurification. It can have a high partition coefficient for theprecursor. The solvent can contain liquid(s) other than toluene itself.Those other liquid(s) are, however, preferably solvents for the compoundas well. Specifically, the invention contemplates the use of acetone inthe solvent and hence the solvent preferably comprises toluene andacetone. However, if other liquid(s) are employed, then preferably thetoluene is present at least 50%. Alternatively, instead of being presentin the solvent, the acetone can be added directly to the composition: inthis case it is prefered to have a composition:acetone ratio of from5:95 to 70:30 (by volume). Whichever way the acetone is added,preferably the ratio (by volume) of toluene:solution is between 1:1 and5:1. If the process of the second aspect follows the first, then theeluate will already contain some acetone. Lactonization in (c) can thentake place, suitably in the same solvent that was used for extraction.

Lactonization of a compound of formula I, to produce a compound ofgeneral formula II, can be achieved by heating in the solvent (which canbe only toluene). The heating can be from 80° C. to 110° C., e.g. 90° C.to 100° C. (usually under atmospheric pressure). Most of any water oracetone present in the solvent can thus be removed by evaporation atthis temperature. Preferably, heating is continued for at least 6 hours.However, heating is preferably not beyond 130° C. as by-products, suchas a lovastatin dimer, may be formed.

If the solvent contains acetone, then most of this will have evaporatedduring the heating process although it is not imperative that allacetone is removed. At preferred conditions, one can obtain a acetoneconversion of 98% or higher.

After heating, the solvent is allowed to cool, suitably to roomtemperature.

Lactonization is an equilibrium reaction: in effect, one is performingring closure, where the by-product is water which may then evaporateduring the heating process.

A third aspect of the invention (which may follow the process of thesecond aspect) provides a process of purifying (or isolating) compoundof general formula II (which may be HMG-CoA reductase inhibitor) from acomposition comprising the compound and toluene, the process comprising:

(a) contacting the composition with a (first) aqueous solvent and, ifnecessary, adjusting the pH to from 8 to 11, optionally followed byremoving at least part of the aqueous solvent;

(b) either before or after (a), contacting the (toluene-containing)composition with a (second) aqueous solvent and, if necessary, adjustingthe pH to from 2 to 6, and optionally removing at least part of theaqueous solvent; and

(c) further purifying, or isolating, the compound from the resulting(toluene-containing) composition.

In effect, therefore, (a) and (b) can be thought of as two extractionseach with a respective aqueous solvent, the first at a pH of from 8 to11 and the second at a pH of from 2 to 6. Although the (alkaline)aqueous wash in (a) has been placed first, in practice they can beinterchanged so that (b) is performed first followed by (a).

The process of the third aspect can be subsequent to the process of thesecond (and additionally the first) aspects. Preferably the compound isone that is to be administered as an inhibitor, and is of the generalformula II

wherein:

each of R¹ and R² independently represent a hydrogen atom, a methylgroup or a hydroxyl group; and

R³ represents a (straight chain or branched) C₂₋₆ alkyl group;

and there is one double bond present in the first ring, between eithercarbon atoms 3 and 4 or atoms 4 and 4a, and no or one double bond in thesecond ring, which if present is between either carbon atoms 4a and 5 oratoms 5 and 6.

Three preferred compounds (all of formula II) have the followingformulae:

Also of formula II is simvastatin which has the same structure aslovastatin except that the 1-methyl propyl side chain is replaced by a1,1-dimethyl propyl group.

The third aspect can thus involve two (aqueous) washings of thecomposition containing the toluene and the desired compound. Thesewashings with aqueous solvents can remove undesirable components: thus,unwanted components that are water-soluble, can be extracted into theaqueous solvents. Since the solvents are aqueous, they will not bemiscible with the toluene-containing composition. The toluene-containingcomposition and aqueous solvent can therefore be mixed, and (the two)phases allowed to separate. In both (a) and (b) at least part (andpreferably all) of the aqueous solvent is removed following mixingand/or stirring with the toluene-containing composition.

In stage (a) components with a polar character at a pH from 8 to 11 canbe removed, for example any remaining compounds of formula I (such aslovastatin in its acid form). Polar components may remain in thetoluene-containing composition following lactonization. Components thatremain unconverted as a result of the lactonization process can therebybe removed. Preferably the pH is not above 11, otherwise compounds offormula II may be reconverted back into their open-ringed form (offormula I).

In stages (a) and (b), the aqueous solvent can first be added, beforethe pH is adjusted, or this performed the other way around. The first ispreferred (that is to say, the aqueous solvent is added first, before pHadjustment) otherwise an emulsion can form (of the type water-in-oilsuch as where the aqueous solvent exists as particles within thetoluene-containing composition as the continuous phase).

As will be appreciated, pH adjustment is not necessary if the aqueoussolvent is at the correct and desired pH. In (a), this can be achievedby any suitable alkali, for example an alkali metal hydroxide, such assodium hydroxide (e.g. at about 2N). In (b), any suitable acid can beemployed, but it is preferably a mineral acid, such as hydrochloricacid, nitric acid or (preferably) sulphuric acid.

The composition is, in preferred embodiments, contacted with the aqueoussolvent in (a) and/or (b) in an amount to give a ratio (by volume) offrom 2-3:1 toluene composition:water. The pH in (a) is suitably from 8to 11, preferably from 9 to 10 and most preferably about 10. The twophases are suitably mixed for from 5 to 60 minutes, preferably from 20to 40 minutes, and then separated from each other.

A second quantity (e.g. of water) can then be added to (then separated)toluene-containing layer. In (b) the pH is preferably from 2 to 6, suchas from 3 to 5 and optimally about 4. The phases are suitably mixed forfrom 5 to 60 minutes, preferably from 20 to 40 minutes, and thenseparated from each other.

The compound of formula II can be further isolated and purified bystandard techniques known in the art. For example decolorization can beachieved by using activated carbon although one can use any decolorizingagent known in the art. This may remove any coloured and/or hydrophobiccomponents (such as from the resulting toluene-containing composition).Decolorization may be preferred if the reductase inhibitor is of a lightcolour, for example it is white. However, if the process of the thirdaspect follows on from the process of the first and second aspects, thenthe treatment with the resin in (c) of the first aspect may have alreadyremoved some of the colouring components.

Crystallization can be carried out using standard techniques and furtherpurification can be achieved by, for example, recrystallisation orchromatography.

A preferred process of the invention, which involves all three aspects,therefore comprises a process for preparing a compound of formula I orII:

wherein:

each of R¹ and R² independently represents a hydrogen atom, a methylgroup or a hydroxyl group; and

R³ represents a (straight chain or branched) C₂₋₆ alkyl group;

or a salt or isomer thereof;

and there is one double bond present in the first ring, between eithercarbon atoms 3 and 4 or atoms 4 and 4a, and no or one double bond in thesecond ring, which if present is between either carbon atoms 4a and 5 oratoms 5 and 6;

the process comprising:

providing a composition comprising cells that have produced compound offormula I or II; and

(1) adjusting, if necessary, the pH of the composition to be at least7.5;

(2) removing the cells from the composition to obtain a solution of thecompound;

(3) optionally heating the solution;

(4) contacting the solution with a resin so that the compound isadsorbed onto the resin, removing the compound from the resin into anaqueous composition arid, if necessary adjusting the pH of thecomposition to be less than 7;

(5) contacting the composition with a water-immiscible solventcomprising toluene, so that at least some of the compound is extractedby dissolving in the (toluene-containing) solvent;

(6) if the compound is of formula I, optionally lactonizing the compoundin the solvent to produce a compound of formula II;

(7) contacting the resulting composition comprising the compound andtoluene with a (first) aqueous solvent and, if necessary, adjusting thepH to be from 8 to 11, and optionally removing at least part of theaqueous solvent;

(8) either before or after stage (7), contacting the(toluene-containing) composition with a (second) aqueous solvent and, ifnecessary, adjusting the pH to from 2 to 6, and optionally removing atleast part of the aqueous solvent;

(9) optionally decolorizing the resulting (toluene-containing)composition;

(10) optionally performing crystallization and/or drying, and if desiredfurther purifying the compound.

As will be appreciated, the preferred features and characteristics forthe first, second and third aspects can be incorporated into theprotocol outlined in the previous paragraph.

One advantage of the process according to the present invention comparedto prior art extraction processes can be that the use of a resin (foradsorption/desorption) can reduce the amount of solvent required. Theyield of compound obtained can be high, despite the number of stepsinvolved in the process. It should be borne in mind that as the threeaspects (processes) of the invention can be performed, in that order, aspart of or to form a larger multi-stage process, preferred features ofone aspect, where applicable, are valid for another aspect mutatismutandis.

The following examples are provided to illustrate and in no way limitthe scope of the process of the present invention and should not beconstrued as being limiting.

Comparative Example 1

Production of Lovastatin Containing Fermentation Broth of AspergillusTerreus

Aspergillus terreus strain AD43, DS number 28373 has been deposited withthe Centraal Bureau voor Schimmelcultures (CBS, Delft, The Netherlands)and has been granted CBS accession number CBS 456.95.

One 1 ml vial of a spore suspension of Aspergillus terreus strain AD43,stored in glycerol at −80° C. was opened aseptically, and contents weresuspended in a 2 liter shake flask containing 500 ml of the followingmedium (heated in an autoclave for 20 minutes at 121° C.).

Ingredient Amount per kg Glucose.1H₂O 10 g Oatmeal 10 g Tomato paste 40g Corn steep liquor 5 g Trace elements 1 g

The composition of the trace element solution (per 100 ml of distilledwater) was: FeSO₄.7H₂O, 1 g; MnSO₄.1H₂O, 1 g; CuCl₂.2H₂O, 0.025 g;CaCl₂.2H₂O, 0.1 g; H₃BO₄, 0.056 g; (NH₄)₆Mo₇O₂₄.4H₂O, 0.019 g;ZnSO₄.7H₂O, 0.2 g.

The shake flasks were incubated at 28° C. for 24 hours in a rotaryshaker at 280 rpm (throw of 3.5 cm). Eight flasks were used forinoculation of one fermenter with 2000 kg of broth weight. Thecomposition of the fermentation broth was as follows:

Ingredient Amount per kg Glucose.1H₂O 22 g Yeast extract paste 33 gPolypropylene glycol 2000 2.5 ml

Before sterilisation the pH of the solution was decreased to pH 4.5 withsulphuric acid. Sterilisation was performed for 45 minutes at 121° C.Fermentation conditions were as follows: pH was kept constant at 6.5using H₂SO₄ and NaOH; temperature was 28° C.; dissolved oxygenconcentration was kept at 20% by stirring speed, overpressure and airsupply. Minimum values were respectively: 150 rpm, 0.2 bar and 155 kg ofair per hour.

As soon as all the glucose was consumed a glucose/yeast extract feed wasstarted at a rate of 1.2 g of glucose per kg of broth per hour. Thecomposition of the feed was:

Ingredient Amount per kg Glucose.1H₂O 550 g Yeast extract paste 17 gPolypropylene glycol 2000 14 ml

After 192 hours this fermentation yielded 750 mg lovastatin per liter.

EXAMPLE 2

Isolation of Crude Lovastatin Crystals Via Extraction of Broth Filtrate

The pH of 1.8 L of broth of strain AD43, produced according to Example1, was adjusted to pH 10. After half an hour the broth was filtered andthe filter cake was washed with three cake volumes of water, resultingin 3 L of broth filtrate with a lovastatin acid concentration 0.40 g/l.

The pH of the filtrate was brought to pH 12.5 with 2 N NaOH at 25° C.,and subsequently heated at 50° C. for 2 hours. After 2 hours thereaction was complete, and the reaction mixture cooled to roomtemperature. The pH was lowered to pH 4 using sulphuric acid and 9 L oftoluene were added and mixed over 30 minutes. The toluene layer wasseparated from the water layer and subsequently concentrated to a volumeof 250 ml by evaporation at 40° C. under vacuum to give a lovastatin(acid) concentration of 4.3 g/l.

The lovastatin acid in the extract was converted into the lactone byheating it to 90° C. for 3 hours (yield of conversion was 99.2%). Aftercooling to room temperature, the toluene was mixed with 250 ml of water,while the pH was adjusted to pH 10 with NaOH. After separation of thelayers, the toluene layer was mixed again with 250 ml of fresh water,while the pH is adjusted to pH 4 with sulphuric acid. After separationof the layers, the toluene layer was treated with 0.3 g of activatedcarbon, Norit SX Ultra™.

Subsequently the toluene solution was filtered and further concentratedto 15 ml by evaporation. Cooling to −10° C. resulted in crystallization.The crystals were washed with 5 ml of cold toluene, and dried undervacuum at room temperature, yielding 0.83 g of crude lovastatin crystalswith a purity of 90.7% (HPLC).

EXAMPLE 3

Isolation of Lovastatin from Fermentation Broth Via Extraction of BrothFiltrate

Two fermentations of 2.5 m³ each, as performed in Example 1, werecombined. The pH was brought to pH 10 with 2 N NaOH and stirred for 0.5hr. The broth was filtered over a membrane filter press, yielding 3100 Lof filtrate with 0.54 g of lovastatin.

2100 L of broth filtrate was extracted in three subsequent portions of700 L each with 2100 L of toluene, in order to obtain a good phaseseparation. The overall extraction yield was 90%. The extract wasconcentrated by evaporation of toluene to a lovastatin (acid)concentration of 5 g/l (final volume 200 L), and then the lovastatinacid in the extract lactonized by heating the solution at 90° C. for 6hr (to give a conversion of 97.6%).

After cooling to room temperature, 200 L of water was added, whereafterthe pH was adjusted to pH 10 with 2 N NaOH. After 0.5 hr the phases wereseparated, and another 200 L of water added to the toluene phase,whereafter the pH was adjusted to pH 4 with 2N H₂SO₄. After 0.5 hrmixing, the phases were allowed to settle and separate. Then 300 g ofactive carbon Norit SX-Ultra™ was added to the toluene layer, mixed for1 hour and filtered.

The toluene phase was further concentrated to a lovastatin concentrationof 60 g/l by evaporation of toluene at 70° C. under reduced pressure.Then the toluene phase was slowly cooled at a rate of 0.25° C./min to−10° C. The crystals were filtered, washed with 3 L of cold toluene(−10° C.) and dried at room temperature under vacuum, yielding 760 g ofcrude lovastatin crystals with a purity of 93.5% (HPLC).

EXAMPLE 4

Isolation of Crude Lovastatin Crystals Using Hydrophobic Adsorption ofLovastatin Acid from Broth Filtrate

The pH of 15 L of broth of strain AD43, which was produced according toExample 1, was adjusted to pH 10 with 2N NaOH. After 30 minutes stirringat pH 10, the broth was filtered, and the filter cake was washed with 3cake volumes of water. The filtrate (lovastatin acid concentration 0.40g/l) was brought to pH 12.5 with 2N NaOH at 25° C., and subsequentlyheated at 60° C. for 30 min. After 30 minutes the reaction was complete,and the reaction mixture was cooled to room temperature and filtered,resulting in 25 L of broth filtrate with a lovastatin acid concentrationof 0.43 g/l.

A column was prepared with 300 ml of the hydrophobic resin XAD-16™.Prior to loading the filtrate onto the column, the resin was conditionedwith 0.02 N NaOH. Subsequently, the pH of the filtrate was adjusted topH 12, and lovastatin acid adsorbed onto the hydrophobic resin XAD-16 byleading 25 L of the heat-treated filtrate over the column. The columnwas washed with 3 bedvolumes of 0.2 N NaOH. Lovastatin acid wassubsequently eluted from the resin with 3 bedvolumes of a mixture of 70%acetone in water. The concentration of lovastatin acid in the eluate was11 g/l.

Subsequently, the eluate was extracted with 2 L of toluene at pH 4.After phase separation, acetone and water were evaporated at atemperature of 110° C., the toluene-solution was then kept attemperatures between 90° C. and 110° C. to induce lactonization. After 6hours of reaction time, 98% of lovastatin acid was converted intolovastatin. The solution was cooled to room temperature and washed withwater by adding 1 L of water to the toluene. The pH was adjusted to pH10 with 2 N NaOH. The phases were separated, and the washing procedurerepeated once more. Afterwards, 1 L of water was again added to thetoluene phase, and the pH adjusted to pH 4 with 2 N H₂SO₄. Afterseparation of the phases, 7 g of Norit SX-Ultra™ was added, and stirredfor 1 hour before filtration.

The toluene was then evaporated to obtain a lovastatin concentration of60 g/l. The solution was cooled at a rate of 0.5° C./min to −10° C.,yielding 7.4 g of crude lovastatin crystals with a purity of 92.2%.

EXAMPLE 5

Isolation of Lovastatin Acid Via Hydrophobic Adsorption of LovastatinAcid from Broth Filtrate

To 2.5 m³ of broth of strain AD43, produced according to Example 1, 1250L of water was added. Subsequently the pH of the diluted broth wasadjusted to pH 10 with 2 N NaOH. After 30 minutes stirring at pH 10, thebroth was filtered, yielding 3 m³ of filtrate with a lovastatin acidconcentration of 0.44 g/l. Subsequently the pH of the filtrate wasbrought to pH 12.5 with 2 N NaOH at 25° C., and then was heated at 60°C. for 30 min. After 30 minutes, the reaction was completed, and thereaction mixture was cooled to room temperature and filtered, resultingin 3 m³ of broth filtrate with a lovastatin acid concentration of 0.47g/l.

A column was prepared with 20 L of the hydrophobic resin XAD-16™. Priorto loading the filtrate onto the column, the resin was conditioned with0.02 N NaOH. Subsequently, the pH of the filtrate was adjusted to pH 12,and lovastatin acid was adsorbed onto the hydrophobic resin XAD-16 byleading 1.5 m³ of the heat-treated filtrate over the column. The columnwas washed with 3 bedvolumes of 0.2 N NaOH. Lovastatin acid wassubsequently eluted from the resin with 3 bedvolumes of a mixture of 70%acetone in water. The concentration of lovastatin acid in the eluate was11.5 g/l.

Subsequently, the eluate was extracted with 140 L of toluene at pH 4.After phase separation, acetone and water were evaporated at atemperature of 110° C., the toluene-solution (lovastatin content 4.7g/1) was then kept at temperatures between 90° C. and 110° C. to inducelactonization. After 6 hours of reaction time, 99% of lovastatin acidwas converted into lovastatin. The solution was then cooled to roomtemperature and washed with water by adding 70 L of water to thetoluene. The pH was adjusted to pH 10 with 2 N NaOH. The phases wereseparated, and the washing procedure repeated once more. Again 70 L ofwater was added to the toluene phase, and the pH adjusted to pH 4 with 2N H₂SO₄. After separation of the phases, 200 g of Norit SX-Ultra™ wasadded, and stirred for 1 hour before filtration.

The toluene was then evaporated to obtain a lovastatin concentration of60 g/l. The solution was cooled at a rate of 0.25° C./min to −5° C.,yielding 530 g of crude lovastatin crystals with a purity of 95.5%(HPLC).

EXAMPLES 6 & 7

Comparison of the Extraction of Lovastatin from Broth Filtrate withToulene with and without the Presence of Acetone

The pH of 100 ml of a broth filtrate, obtained from the fermentationbroth of Example 1, containing 1 g/l of lovastatin was adjusted to pH 4with sulphuric acid. The broth filtrate was mixed with an equal amountof toluene, and stirred for 15 minutes. After separation of the emulsionby centrifugation, the toluene phase (93 ml) had a lovastatin content of0.6 g/l, which is a yield of 58% (Example 6).

The pH of another 100 ml of the same fermentation broth filtrate wasalso adjusted to pH 4 with sulphuric acid. Then (Example 7) a mixture of85 ml of toluene and 15 ml of acetone were mixed with the broth over aperiod of 15 minutes. After separation, the toluene layer (99 ml) had alovastatin content of 1.13 g/l, a yield of 100%.

What is claimed is:
 1. A process for recovering from a fermentation broth a compound having formula I or II:

or a salt or isomer thereof; wherein: each of R¹ and R² independently represents a hydrogen atom, a methyl group or a hydroxyl group; and R³ represents a C₂₋₆ alkyl group; and there is one double bond in the first ring between either carbon atoms 3 and 4 or 4 and 4a, and no or one double bond in the second ring between either carbon atoms 4a and 5 or 5 and 6; the process consisting essentially of: (1) adjusting the pH of the fermentaion broth to be at least 7.5 therby solubilizing any compound from the cells; (2) removing the cells from the pH-adjusted fermentation broth to obtain a solution of the compound; (3) heating and/or adjusting the pH of the solution to convert unwanted byproduct into said compound; (4) contacting the solution of step (4) with a resin to absorb the compound onto the resin, eluting the compound from the resin into forming an aqueous composition and, if necessary adjusting the pH of the composition to be less than 7; (5) contacting the aqueous composition with a water-immiscible solvent comprising toluene to extract the compound; (6) lactonizing any compound having formula I in the extract solvent to form compound having the formula II; (7) contacting the extract solvent comprising the compound and toluene with a first aqueous solvent and, if necessary, adjusting the pH to be from 8 to 11, and optionally removing at least part of the aqueous solvent; (8) either before or after stage (7), contacting the toluene-extract solvent with a second aqueous solvent and, if necessary, adjusting the pH to from 2 to 6, and optionally removing at least part of the aqueous solvent; (9) decolorizing the resulting toluene-containing extract-solvent; and (10) recovering the compound through crystallization and/or drying the compound.
 2. A process according to claim 1 wherein in step (1) the pH of fermentation broth containg the cells is adjusted to a pH of from 9 to 13 and the resin in step (4) is hydrophobic.
 3. A process according to claim 1 wherein in step (4) the compound is removed from the resin by elution with a water-miscible solvent comprising acetone and the resin is a polystyrene resin.
 4. A process according to claim 1 wherein in step (4) the pH of the aqueous composition is adjusted to from 3 to 5 and the solvent in step (5) comprises toluene and acetone.
 5. A process according to claim 1 wherein in step (6) the compound having formula (1) is lactonized by heating at from 80° C. to 120° C., optionally directly in the solvent from step (5).
 6. A process according to claim 1 wherein the pH in step (7) is from 9 to 10 and the pH in step (8) is from 3 to
 5. 7. A process according to claim 1, wherein the compound is lovastatin, pravastatin or compactin.
 8. A process of purifying from a fermentaiton broth containing cells that produced a compound of the general formula I or II:

or a salt or isomer thereof; wherein: each of R¹ and R² independently represents a hydrogen atom, a methyl group or a hydroxyl group; and R³ represents a C₂₋₆ alkyl group; and there is one double bond present in the first ring, between either carbon atoms 3 and 4 or 4 and 4a, and no or one double bond in the second ring, which if present, is between either carbon atoms 4a and 5 or 5 and 6; the process consisting essentially of: (a) adjusting the pH of the fermentaiotn broth containing the cells with an akali so that the broth is at least 7.5 therby solubilizing any compound from the cells; (b) removing the cells from the fermentation broth to obtain a solution of the compound; (c) contacting the solution with a resin to absorb the compound onto the resin; and (d) removing the compound from the resin.
 9. A process according to claim 8 wherein the compound is lovastatin, pravastatin or compactin. 